Baculovirus expression system expresses hplgf-1 strongly
,1,* Mahdi behdani
1. Infectious Diseases and Tropical Medicine Research Center, Resistant Tuberculosis Institute, Zahedan University of Medical Sciences, Zahedan, Iran.
2. Biotechnology Research Center, Venom & Biotherapeutics Molecules Lab, Pasteur Institute of Iran, Tehran, Iran.
Baculovirus expression system has been used successfully to over express eukaryotic proteins in insect cells. this system uses a very strong viral promoter, acnpv polyhedrin, for high level of protein expression. baculovirus system has brought advantages, such as fast growth ability, high density in simple and inexpensive environments, genetics and specific molecular biology, high rates of expression of recombinant protein. angiogenesis is the most important factor in physiological and pathological conditions. human placental growth factor (hplgf-1) protein is a member of the vegf family, which plays an important role in angiogenesis. hplgf-1 binds to vascular endothelial growth factor receptor-1(vegfr1) or flt1 (fms-like tyrosine kinase-1). hplgf-1 plays a role in the differentiation and growth of the fibroblast, and playing various roles in pathological angiogenesis which occurs in tissue ischemia, inﬂammation, and malignancy, with its plasma level associated with the stage and the prognosis of cancers.
in this report, we evaluated the efficiency of baculovirus as a shuttle vector for delivery of hplgf-1gene into insect cells and produce high levels of protein.
Hplgf-1gene cloned in pfastbac-ht vector and transformed in dh10bac. sf9 cells were grown in grace’s medium with 1% penicillin-streptomycin and 10% fetal bovine serum and incubated in 27°c .the recombinant bacmid was extracted and used in sf9 insect cells and trasfected by cellfectin method. target protein expression was confirmed by sds-apge and western blotting.
Transferring of the recombinant vector into bacmid was successful and the hplgf-1 gene sequences were confirmed. the recombinant bacmid dna was then generated by transfection of sf9 insect cells using cellfectin® reagent and propagated in sf9 insect cells using the liposome mediated method. ten days after transfection, cytopathic signs were observed and the size of cells was increased and was gradually lysed. hplgf-1 and recombinant protein expression by western blotting was confirmed. it clearly demonstrated a band with a molecular weight of 60 kda.
The protein was produced at high level and was localized to the expected position in the cell. these two features are the basic characteristics of an appropriate expression system. baculovirus protein expression system expresses hplgf-1 and recombinant protein can be used in different tests.
Baculovirus expressive system, hplgf-1, angiogenesis